Gamma-glutamyl transpeptidase (GGT) activity is commonly used to follow the differentiation of liver precursor cells into the biliary lineage. However, the GGT expression in immature hepatocytes or its induction in adult hepatocytes following diverse carcinogenic or noncarcinogenic treatments has questioned the reliability of GGT expression as a biliary marker. In the present study, we investigated the GGT gene expression from its five different promoters in the late fetal, neonatal, and adult rat liver by Northern blot, reverse transcription-polymerase chain reaction, and in situ hybridization analysis. We show that the GGT activity in the 18-day-old fetus results from the transcription of the gene from the promoter P3 in the hepatocytes. In contrast, the GGT promoter P4 activity appears to be specific of biliary cells in normal as well in cholestatic liver. Thus, sequences unique to the GGT transcripts initiated on these two alternate promoters provide unique molecular probes to discriminate between the biliary and the hepatocytic phenotypes in liver differentiation and cell lineage studies.